T-cell subsets include helper T-cells (CD4+) and cytotoxic T-cells (CD8+ CTL). CD4+ helper T cells can generally be broken down into several different subtypes. Commonly divided populations include T helper 1 (Th1) and T helper 2 (Th2) cells which have been found to play distinct roles in mediating immune-related diseases and disorders. Th1 and Th2 cells can be distinguished based on their cytokine expression profile. Th1 cells typically express interferon-gamma (IFN-g, IFN-γ), lymphotoxin, (LT), and can also secrete interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-a, TNF-α) (Zhu et al., Annu Rev Immunol. 28: 445-489, 2010). Th2 cells are characterized by production of IL-4, IL-5 and IL-13, and do not secrete IFN-γ or lymphotoxin. Some Th2 cells have been found to produce TNF-α and IL-9 (Zhu, supra). Th17 cells have also recently been determined to be a different subset of T helper cells. Th17 cells secrete IL-17A, IL-17F, and IL-22 cytokines, and can also produce IL-21. The dysregulation of the ratio of Th1 to Th2 cells, as well as Th17 cells, has been associated with certain disease states, including autoimmune diseases, allergic reactions and cancers. Two additional CD4+ T-cell subsets that can be identified by cytokine production include Treg (IL-10) and follicular helper T cells (Tfh) (IL-21).
Polyfunctional, CD8+ CTL are important effector cells that provide protection from intracellular pathogens and tumors where polyfunctionality is defined as production of multiple cytokines including (e.g. IFN-g, TNF-a and/or IL-2). In cancer subjects these polyfunctional T-cell responses have been shown to be repressed. Several new treatments (e.g. anti-CTLA4, anti-PD1) have been shown reverse functional suppression of CTL and combination with TVEC (talimogene laherparepvec) holds further promise for increasing the number of functional, activated CTL that can contribute to meaningful anti-tumor responses. Additional T cell subsets include natural killer T cells (NKT) and gamma delta T cells (gdT).
Determining levels of T cells in vivo and determining the levels of cytokines produced by these cells can be difficult. Recent advances in laboratory techniques have provided several methods for determining T helper subtypes and cytokine levels, including ELISA, ELISPOT, and flow cytometry assays, including intracellular cytokine staining assays. Despite the advancement in methodology, it is still difficult to identify particular T cell subtypes and their cytokine profiles in a patient with a disease or disorder because the levels of these particular cell types and cytokines in a given patient sample can be low to undetectable.
A common technique to identify and separate one cell type from another is flow cytometry using labeled dyes, e.g., in a fluorescence activated cells sorter (FACS). Methods of carrying out flow cytometry are discussed, for example, in U.S. Pat. No. 8,389,291, U.S. Pat. No. 7,932,503, U.S. Pat. No. 7,012,689, and U.S. Pat. No. 6,287,791.
In addition to cytokines, combinations of cell surface markers can also be used to classify T-cells by subset (e.g. Th2 cells express CRTH2) or activation status (e.g. activated CTL express HLA-DR) by FACS.
Previous studies have undertaken experiments to analyze T helper cell profiles in response to stimuli. International Patent Publication WO 1997/026883 describes intracellular cytokine staining for IL-2 only on PBMC stimulated in vitro with PMA/ionomycin with or without Ribavirin®. The results provided describe a putative pharmacological effect based on in vitro data.
U.S. Pat. No. 6,039,969 describes animal model data demonstrating that a class of compounds including the drug imiquimod can skew the immune response away from Th2, based primarily on secreted cytokine results. Human clinical data and intracellular cytokine staining were not disclosed.
U.S. Patent Publication 2001/0006789 describes a method used to identify antigen specific T-cells. Measurement of pharmacological effects on antigen specific T-cells is not demonstrated.
International Patent Publication WO 2000/024245 describes a method for targeting NFATp and/or NFAT4 to modulate Th2 cells. WO 2002/089832 relates to a combination of cytokine+SDF-1a to skew the Th1/Th2 ratio. In vitro data, including intracellular cytokine staining specific for IFN-g and IL-4, using cultured cord blood T-cells are presented. Human clinical data demonstration is not included.
Lore et al. (J Immunol 171:4320-28, 2003) describes analysis of CMV- and HIV-specific CD4+ and CD8+ T cells using a method in which levels of the Th1-specific cytokines IFN-g, TNF-a and IL-2 are measured in a single sample by flow cytometric analysis. The Th1 cytokines are detected using cytokine-specific antibodies, each of which is labeled with the same fluorophore. This method does not determine the ratio of Th1 to Th2 cells.
Ludviksson et. al. (J Immunol 160:3602-3609, 1998) describes association of Wegener's Granulomatosis with HLA-DR+CD4+ cells exhibiting unbalanced Th1 cell cytokine pattern and reversal with IL-10. Wang et. al. (Int Imm 21:1065-1077, 2009) demonstrate that PD1 blockade reverses functional suppression of CTL in melanoma setting. Sfanos et. al. (Clin Cancer Res 14:3254-3261, 2008) demonstrate skewing of tumor infiltrating lymphocytes to Th17 and Treg subsets in prostate cancer.
The present disclosure is directed to methods for identifying and differentiating a population of T helper or CTL cells in a subject having a disease or disorder with an immune component. The present technique can be carried out before and after treatment with a therapeutic agent to improve treatment regimens for patients receiving therapy.